U50,488H Reduces Ischemia-reperfusion Induced Cardiomyocyte Apoptosis And The Underlying Mechanism

AIMS(1) To investigate the effect of U50,488H, a specificκ-opioid agonist, on I/R induced cardiomyocyte apoptosis in rat heart.(2) To investigate the effect of U50,488H on PI3K-Akt-eNOS-NO pathway in I/R injured rat heart.(3) To determine the role of PI3K-Akt-eNOS-NO signaling pathway in U50,488H mediated anti-apoptotic effect in I/R injured rat heart.METHODSMale Sprague-Dawley rats were subjected to 30 minutes of myocardial ischemia and 3 hours of reperfusion. Rats were randomized to receive either vehicle, U50,488H, U50,488H plus Nor-BNI, a selectiveК-opioid receptor antagonist, U50,488H plus wortmannin, a specific inhibitor of phosphoinositide 3'-kinase (PI3K), or U50,488H plus L-NAME, a nitric oxidase synthase inhibitor (NOS inhibitor), immediately prior to reperfusion. The infarction area, the apoptotic ratio of cardiomyocyte, the activity of Caspase-3, the total amount and phosphorylation of Akt and eNOS and the production of NO were examined respectively. In vitro study was performed on cultured neonatal cardiomyocytes subjected to simulated ischemia/reperfusion. Cultured neonatal cardiomyocytes were randomized to receive either vehicle, U50,488H, U50,488H plus Nor-BNI, U50,488H plus wortmannin, U50,488H plus L-NAME, or U50,488H plus specific Akt inhibitor in simulated reperfusion after subjected to simulated ischemia. The apoptotic ratio of cardiomyocyte, the activity of Caspase-3, the total amount and phosphorylation of Akt and eNOS and the production of NO were examined respectively.RESULTSTreatment with U50,488H resulted in 1.7-fold and 2.6-fold increases in Akt and endothelial nitric oxidase synthase (eNOS) phosphorylation and a significant increase in nitric oxide (NO) production in ischemic/reperfused myocardial tissue. Phosphorylation of Akt and eNOS and increase in NO production by U50,488H were completely blocked by wortmannin. Pretreatment with L-NAME, had no effect on Akt and eNOS phosphorylation but significantly reduced NO production. Moreover, treatment with U50,488H markedly reduced myocardial apoptotic death (P<0.01 versus vehicle). Pretreatment with wortmannin abolished the anti-apoptotic effect of U50,488H. Most interestingly, pretreatment with L-NAME also significantly attenuated the anti-apoptotic effect of U50,488H (P<0.01 versus U50,488H). All the anti-apoptotic effects of U50488H were blocked by nor–BNI, a selectiveК-opioid receptor antagonist. In cultured neonatal cardiomyocytes subjected to simulated ischemia/reperfusion (SI/R), U50,488H increased Akt activity and eNOS phosphorylation with subsequent NO production, and concurrently exerted an anti-apoptotic effect, all of which are abolished by not only Nor-BNI, wortamnnin, L-NAME but also by the specific Akt inhibitor.CONCLUSION(1) U50.488H reduces I/R injury induced cardiomyocyte apoptosis in rat in vivo.(2) U50,588H increases Akt and eNOS phosphorylation and NO production and by doing so activates the PI3K-Akt-eNOS-NO pathway.(3) The anti-apoptotic effect of U50,488H is at least partially mediated by the PI3K-Akt-eNOS-NO signaling pathway.